The developments of methods for nucleic acid amplification and detection of amplification products have advanced the detection, identification, quantification and sequence analyses of nucleic acid sequences in recent years.
Nucleic acid analysis is useful for detection and identification of pathogens, detection of gene alteration leading to defined phenotypes, diagnosis of genetic diseases or the susceptibility to a disease, assessment of gene expression in development, disease and in response to defined stimuli, as well as the various genome projects. Other applications of nucleic acid amplification method are the detection of rare cells, detection of pathogens, and the detection of altered gene expression in malignancy, and the like. Nucleic acid amplification is potentially useful for both qualitative analysis, such as the detection of the presence of defined nucleic acid sequences, and quantification of defined gene sequences. The latter is useful for assessment of and amount of pathogenic sequences as well as the determination of gene multiplication or deletion, as often found in cell transformation from normal to malignant cell type.
The detection of sequence alterations in a nucleic acid sequence is important for the detection of mutant genotypes, as relevant for genetic analysis, the detection of mutations leading to drug resistance, pharmacogenomics, etc. Various methods for the detection of specific mutations include allele specific primer extension, allele specific probe ligation, and differential probe hybridization. See, for example, U.S. Pat. Nos. 5,888,819; 6,004,744; 5,882,867; 5,710,028; 6,027,889; 6,004,745; and WO US88/02746. Methods for the detection of the presence of sequence alterations in a define nucleic acid sequence, without the specific knowledge of the alteration, were also described. Some of these methods are based on the detection of mismatches formed by hybridization of a test amplification product to a reference amplification product. The presence of mismatches in such hetero-duplexes can be detected by the use of mismatch specific binding proteins, or by chemical or enzymatic cleavage of the mismatch. A method for detection of sequence alteration which is based on the inhibition of branch migration in cruciform four stranded DNA structures was recently described. See, for example, Lishanski, A. et al. Nucleic Acids Res 28(9):E42 (2000). Other methods are based on the detection of specific conformations of single stranded amplification products. The secondary structure of a single stranded DNA or RNA is dependent on the specific sequence. Sequence alterations in a test nucleic acid target relative to a reference sequence leads to altered conformation. Altered conformation of a single stranded amplification product can be detected by a change in the electrophoretic mobility of the test amplification product as compared to that of a reference amplification product. Single stranded conformation polymorphism, SSCP, is widely used for the detection of sequence alterations. See, for example, Orita M., et al. Proc Natl Acad Sci USA 86(8):2766-70 (1989); Suzuki, Y. et al. Oncogene 5(7):1037-43 (1990); and U.S. Pat. No. 5,871,697. This method is also used in microbial identification that is based on the defined changes in a specific nucleic acid sequence in different strains or species. Mutation detection using the SSCP methods mostly employs DNA amplification products, however, RNA-SSCP methods have also been described. Sequence dependent conformation of a single stranded RNA is well-documented and was shown to lead to a defined electrophoretic mobility pattern. See, for example, Sarkar et al. Nucleic Acid Research 20(4):871-878 (1992) and Gasparini et al. Hum. Genet. 97:492-495 (1996).
Although detection of the presence of a defined nucleic acid sequence, and its sequence analysis, can be carried out by probe hybridization, the method generally lacks sensitivity when low amounts of the nucleic acid sequence is present in the test sample, such as a few molecules. One solution to this obstacle was the development of methods for generation of multiple copies of the defined nucleic acid sequence, which are suitable for further analysis. The methods for generation of multiple copies of a specific nucleic acid sequence are generally defined as target amplification methods. Other methods for increasing the sensitivity of detection of hybridization analysis are based on the generation of multiple products from the hybridized probe, or probes, for example cleavage of the hybridized probe to form multiple products or the ligation of adjacent probes to form a unique hybridization dependent product. Similarly, increased sensitivity of hybridization reaction was achieved by methods for amplification of signals generated by the hybridization event, such as the method based on hybridization of branched DNA probes.
There are many variations of nucleic acid amplification, for example, exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification. An example of exponential nucleic acid amplification method is polymerase chain reaction (PCR) which has been disclosed in numerous publications. See, for example, Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Mullis K. EP 201,184; Mullis et al. U.S. Pat. No. 4,582,788; Erlich et al. EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R. et al. U.S. Pat. No. 4,683,194. Linked linear amplification is disclosed by Wallace et al. in U.S. Pat. No. 6,027,923. Examples of ligation-based amplification are the ligation amplification reaction (LAR), disclosed by Wu et al. in Genomics 4:560 (1989) and the ligase chain reaction, disclosed in EP Application No. 0320308B1. Various methods of transcription-based amplification are disclosed in U.S. Pat. Nos. 5,766,849 and 5,654,142; and also by Kwoh et al. Proc. Natl. Acad. Sci. U.S.A. 86:1173 (1989) and Ginergeras et al. WO 88/10315.
The most commonly used target amplification method is the polymerase chain reaction, PCR, which is based on multiple cycles of denaturation, hybridization of two oligonucleotide primers, each to opposite strand of the target strands, and primer extension by a nucleotide polymerase to produce multiple double stranded copies of the target sequence. Many variations of PCR have been described, and the method is being used for amplification of DNA or RNA nucleic acid sequences, sequencing, mutation analysis and others. Thermocycling-based methods that employ a single primer, have also been described. See, for example, U.S. Pat. Nos. 5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. The primer can be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No. 5,744,308. Other methods that are dependent on thermal cycling are the ligase chain reaction (LCR) and the related repair chain reaction (RCR).
Target nucleic acid amplification may be carried out through multiple cycles of incubations at various temperatures, i.e. thermal cycling, or at one temperature (an isothermal process). The discovery of thermostable nucleic acid modifying enzymes has contributed to the fast advances in nucleic acid amplification technology. See Saiki, et al. Science 239:487 (1988). Thermostable nucleic acid modifying enzymes, such as DNA and RNA polymerases, ligases, nucleases and the like, are used in both methods dependent on thermal cycling and isothermal amplification methods. Isothermal methods such as strand displacement amplification (SDA) is disclosed by Fraiser et al. in U.S. Pat. No. 5,648,211; Cleuziat et al. in U.S. Pat. No. 5,824,517; and Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992). Other isothermal target amplification methods are the transcription-based amplification methods, in which an RNA polymerase promoter sequence is incorporated into primer extension products at an early stage of the amplification (WO 89/01050), and further target sequence, or target complementary sequence, is amplified by transcription steps and digestion of an RNA strand in a DNA/RNA hybrid intermediate product. See, for example, U.S. Pat. Nos. 5,169,766 and 4,786,600. These methods include transcription mediated amplification (TMA), self-sustained sequence replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), and variations there of. See, for example, Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878 (1990); U.S. Pat. No. 5,766,849 (TMA); and U.S. Pat. No. 5,654,142 (NASBA). Other amplifications methods use template switching oligonucleotides (TSOs) and blocking oligonucleotides. For example, the template switch amplification in which chimeric DNA primer are utilitized is disclosed in U.S. Pat. No. 5,679,512 and by Patel et al. Proc. Natl. Acad. Sci. U.S.A. 93:2969-2974 (1996) and blocking oligonucleotides are disclosed by Laney et al. in U.S. Pat. No. 5,679,512.
The isothermal target amplification methods do not require a thermocycler, and are thus easier to adapt to common instrumentation platform. However the previously described isothermal target amplification methods have several drawbacks. Amplification according to the SDA methods requires the presence of sites for defined restriction enzymes, which limits its applicability. The transcription base amplification methods, such as the NASBA and TMA, on the other hand, are limited by the need for incorporation of the polymerase promoter sequence into the amplification product by a primer, a process prone to result in non-specific amplification. Moreover, the mechanism of amplification of a DNA target by these transcription based amplification methods is not well-established.
Another drawback of the current amplification methods is the potential contamination test samples by amplification products of prior amplification reactions, resulting in non-target specific amplification in samples. This drawback is a well-known problem, which is the result of the power of target amplification technology, and the formation of amplification products, which are substrates for amplification. Various means for decontamination of test samples either at the end of the amplification reaction, or prior to the initiation of target amplification have been described. In additions, method for containment of the test solution by physical means, were also described. All of these solutions are cumbersome and add to the complexity of nucleic acid testing in the common laboratory setting.
Furthermore, amplification methods that use thermocycling process have an added disadvantage of long lag times which are required for the thermocycling block to reach the "target" temperature for each cycle. Consequently, amplification reactions performed using thermocycling processes require a significant amount of time to reach completion.
Therefore, there is a need for improved nucleic acid amplification methods that overcome these drawbacks. The invention provided herein fulfills this need and provides additional benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.